In the description which follows, references are made to certain literature citations which are listed at the end of the specification and all of which are incorporated herein by reference.
Nuclear receptors are transcription factors involved in various physiological regulatory processes. The superfamily to which nuclear receptors belong comprises both ligand-dependent molecules such as the steroid hormone-, thyroid hormone-, retinoic acid- and vitamin D-receptors, and an increasing number of so-called orphan receptors for which no ligand has yet been determined (Gronemeyer H, Laudet V., 1995; Enmark and Gustafsson, 1996). Indeed, it is not yet known whether the orphan receptors have ligands that await identification or whether they act in a constitutive manner. The orphan receptors display the same structural organization as do the classic ligand-dependent receptors: the A/B domain located in the N-terminal part of the protein harbors a ligand-independent transactivation function (AF-1); the C domain, which is the most conserved part of the molecule, is responsible for the specific DNA-binding activity; the E domain contains the ligand binding hydrophobic pocket and contributes to receptor dimerization and to the ligand-dependent transactivation function (AF-2).
Two orphan receptors, estrogen receptor-related receptor α (ERRα) and ERRβ (Giguere et al., 1988; NR3B1 and NR3B2, respectively, according to the Nuclear Receptors Nomenclature Committee, 1999) are closely related to the estrogen receptors ERα and ERβ (Green et al., 1986; Kuiper et al., 1996; NR3A1 and NR3A2 respectively). ERRα (Genbank Accession No. for human ERRα: NM—004451) and ERRβ were identified by low-stringency screening of cDNA libraries with a probe encompassing the DNA-binding domain of the human estrogen receptor (ER). Recently, a third estrogen receptor-related receptor, ERR3 or ERRγ, was identified by yeast two-hybrid screening with the glucocorticoid receptor interacting protein 1 (GRIP1) as bait (Hong et al, 1999). The DNA binding domain region of ERRs and ERs is highly conserved, however the others parts of the protein share very little homology (Giguere et al, 1988; Hong et al, 1999). Therefore, sequence alignment of ERRα and the ERs reveals a high similarity (68%) in the 66 amino acids of the DNA-binding domain and a moderate similarity (36%) in the ligand-binding E domain, which may explain the fact that ERRα does not bind estrogen. Although ligands for the ERRs have not been clearly identified, the pesticides chlordane and toxaphene have been suggested to be potential ligands for ERRα (Yang and Chen, 1999). ERRα has been identified as a regulator of the SV40 major late promoter during the early-to-late switch of expression (Wiley et al., 1993) and as a regulator of fat metabolism (Sladek et al., 1997; Vega et al, 1997). Yang et al. also showed that ERR modulates the activating effect of estrogens on the lactoferrin promoter and suggested that ERRα may interact with ERs through protein-protein interaction (Yang et al., 1996; Zhang and Teng, 2000). Finally, ERRα has been described as a modulator of the human aromatase gene in breast, and hypothesized to be critical for normal breast development and to play an important role in the pathogenesis and maintenance of breast cancer via its ability to interact with ERs (Yang et al, 1998).
Postmenopausal osteoporosis is a condition caused primarily by the severe decrease of serum estrogen levels after cessation of ovarian function. The absence of estrogen results in an increase in bone turnover (Tumer et al, 1994) and a negative bone remodeling balance, leading to bone loss and an increased fracture risk. An anabolic effect of estrogens on bone homeostasis has been documented in post-menopausal osteoporosis (for review see Pacifi, 1996), where bone loss can be reversed by administration of natural or synthetic estrogens. Although the bone preserving effect of estrogen replacement is indisputable, the molecular and cellular mechanism of action for this hormone effect remain unclear. ERs are expressed in osteoblasts (Turner et al., 1994; Eriksen et al, 1988; Komm et al, 1988), and estrogens have been found to elicit effects ranging from modulation of gene expression to regulation of proliferation in this cell type (for review Harris et al, 1996). In contrast, mice lacking a functional ERα or ERβ have only minor skeletal abnormalities (Korach et al, 1994; Windahl et al, 1999) suggesting that other mechanisms or receptors might be important during skeletal development. ERRβ expression is restricted to early development and to a few adult tissues (Giguere et al., 1988; Pettersson et al., 1996). In contrast, ERRα has a broader spectrum of expression, including fat, muscle, brain, testis and skin (Bonnelye et al, 1997b). Strikingly, ERRα is also highly expressed in the ossification zones of the mouse embryo (in long bones, vertebrae, ribs and skull), and is more widely distributed in osteoblast-like cells than is ERα (Bonnelye et al., 1997a). Moreover it has been shown that ERRα positively regulates the osteopontin gene (Vanacker et al, 1998), an extracellular matrix molecule secreted by osteoblasts and other cells and thought to play a role in bone remodelling among other functions (Denhardt and Noda, 1998).